Abstract Trauma-induced inflammation and multiple organ failure are a major cause of mortality and morbidity in American soldiers and veterans. Gut barrier dysfunction plays a critical role in the development of posttraumatic complications by providing the major site for plasma leakage and bacterial translocation. The intestinal permeability response to burns, a major form of trauma, has not been well characterized, and its cellular and molecular mechanisms remain poorly understood. The goal of this study is to elucidate the cell-specific mechanisms of leaky guts during thermal injury. We propose to characterize the molecular basis of burn-induced hyperpermeability in the intestinal microvascular endothelium and mucosal epithelium. The hypothesis to be tested is that inflammation in the intestinal microcirculation induces endothelial paracellular leakage and neutrophil infiltration into lamina propria leading to mucosal barrier failure. The underlying mechanism involves dynamic remodeling of cell-cell junctions coupled with cytoskeleton contraction triggered by nmMLCK, the non-muscle myosin light chain kinase expressed in endothelial and epithelial cells. The specific aims developed in this proposal are: 1) to characterize the pathophysiology of gut barrier regulation in thermal injury, and 2) to elucidate the molecular mechanisms of gut hyperpermeability. The study design employs complimentary in vivo, ex vivo, and in vitro models that incorporate molecular and genetic approaches into physiological experiments under clinically relevant trauma conditions. A unique aspect of the proposal is that it provides an in-depth evaluation of gut permeability with the ability to discern the endothelial barrier from epithelial barrier function. The significance of this study lies in the potential to establish a new molecular pathway in the regulation of tight and adherens junctions. Data derived from this study would contribute to the advancement of gastrointestinal surgery with implications in the development of effective therapies or surgical interventions against gut barrier injury in patients with trauma and burns. PUBLIC HEALTH RELEVANCE: VA Relevance Trauma is a major cause of mortality and morbidity in American soldiers and veterans. As a typical form of trauma, thermal injury results from flame/chemical burns in combat or anti-terrorist actions as well as from non- work related fire or scald accidents. Despite the improved critical care and wound management, posttraumatic complications remain a life-threatening problem that causes extended hospital stay and charges. The gut plays an important role in the development of multiple organ complications following a severe burn. In particular, gut barrier failure contributes to systemic inflammatory syndrome and sepsis by serving as the major site of blood leakage and bacteria translocation. The mechanisms of leaky guts are poorly understood. This study provides a comprehensive evaluation of the cellular and molecular pathways leading to intestinal hyperpermeability during burn-induced inflammation. The research work has potential impact on the development of surgical or medical interventions against gut barrier injury in VA patients with trauma or inflammatory diseases.